4.8 | Intracellular delivery of Sap
Sap was thiolated by reacting it with 2-iminothiolane hydrochloride (25 eq) for 1.5 h on ice[37,38]. PCNDs were suspended in thiolated Sap solution (2 μM), followed by mechanical rotation in a refrigerator (4 °C) overnight. After washing, Sap-conjugated PCNDs were modified with 9E5-NeutrAvidin conjugates, as described above. DLD1 cells were treated with Sap- and 9E5-conjugated PCND suspension for 3 h. PCND-treated cells were exposed to ultrasound, as described above. After ultrasound exposure, the cells were cultured for 48 h and then stained with calcein-AM. The cells were harvested from the dish using 0.25% trypsin-EDTA and analyzed using a flow cytometer (Guava easyCyte 8; Luminex Japan Co. Ltd., Tokyo, Japan).